Cloning And Expression Analysis Of Perforin Gene Of Carassius Auratus In Qihe River

Perforin (pore forming protein, PFP) is secreted by cytotoxic T lymphocyte (CTL) and natural killercells (NK), and other lymphoid cells. It is a kind of cytoxic protein which has a highly dissolved cellactivity and is not only an important immune effector molecule but also an immunomodulatory molecule.Cytotoxic effect mediated by perforin is the main mechanisms in which T lymphocytes can clear virusinfection and the transformed cells. In this study we have the two kinds of perforin gene named PFP-1andPFP-2from Carassius auratus in Qihe river performed the cloning identification, and use the RT-PCR andQ-PCR technology to analyze the gene expression of them in different tissues and different stage of in earlydevelopment, and also use the Q-PCR technology to analyze the same after the carp is induced by Poly I: Cand PHA as well as acute stressed by Aeromonas hydrophila.The PFP-1and PFP-2were cloned by using degeneracy primers.The PFP-1is with the full cDNAlength of2151bp which contains an ORF of1686bp coding for561aa and contained a5’-UTR of119bpand a3’-UTR of346bp, which weights about63.304kDa and has the pI as9.07,and the PFP·2full cDNAlength is2047bp，including a1674bp ORF which encodes557aa and also contained a5’-UTR of177bpand a3’-UTR of196bp, which weights about60.997kDa and has the pI as9.28. The two PFPs all containMACPF and C2domain which are found in pefforin but do not have transmembrane helix.The results show that PFP-1and PFP-2transcript of the Carassius auratus in Qihe river are expressedin the12taken organizations and the early development phases. In different tissues, PFP-1gene has asignificantly highest expression in muscle, about8times that of the liver. On the contrary, the lowestexpression is in the heart and ovarian. PFP-2gene has a higher expression in gill, about3.5times that of theliver, and the lowest expression is in ovarian. Both perforin gene have a constitutive expression in allexamined organs．In the early development of Carassius auratus in Qihe river, PFP-1gene expressiondeclines during blastopore closed periods with about46times lower than before, and then recover. PFP-2transcription increase was observed in non-fertilized eggs to morula and decrease was observed in crystalsappearing period.In response to Poly I:C treatment，the PFP-1transcription increase was observed in the blood at5d， reaching its peak about1.5times as the control group. But it effects on at3d in the liver and spleen withabout2and3.1times. Finally it reaches the highest in head kidney at day7d with about0.2times. ThePFP-2gene expression in the blood reached the highest in5d after induction, about43times than thecontrol group. The expression quantity is increasing constantly in liver, and reaches its peak in the seventh,which is about9.5times as control group the other in spleen is at7d with about9.5times and in headkidney is at5d with about36times as the control group.In response to PHA injection, PFP-1gene decline is observed in the blood at1d post treatment andthen going up until3d, and PFP-2gene steady expression is at2d. PFP-1and PFP-2gene decline isobserved in liver at1d post treatment; On the other hand, PFP-1and PFP-2gene increase is observed inspleen and head kidney at3d; PFP-2transcription increase is observed in the head kidney，reaching its peakabout43times as the control group.In response to Aeromonas hydrophila acute injection, PFP-1gene decline is observed in allorganizations after6hours,and PFP-2gene decline is observed in the liver、spleen and gill but increase isobserved in kidney、blood and intestines. It shows that: spleen in Aeromonas hydrophila under acute stress,perforin gene expression level decreased, decreased cytotoxicity, may seriously affect the fish immune cellssuch as macrophages, erythrocytes, granulocytes produce, storage and mature release, caused by impairedimmune defense system, eventually leading to the body’s death.